Alternative splicing provides a mechanism for increasing function diversity in eukaryotic organisms. Splice variants (isoforms) are prevalent in the human genome. In the proposed study, the amino acid sequences of isoforms of human genes are mapped onto the three-dimensional structures of homologous proteins that share the same fold. The inferred structural modifications fall into at least six classes. Targets are selected for structural studies on the basis of functional effect of the alternative splicing, involvement of alternative splicing in disease, and the role of structure in mediating alternative function. We will determine the structures of at least 50 isoforms in the course of five years. The goal of the protein production component of the project is to supply sufficient amounts of highly purified protein samples for structural studies. For cases where the functions of the isoforms are still unknown, we will also supply clones and pure proteins to collaborators who will investigate their biological and biochemical functions. Because of the attrition when proceeding from genes to structures, work will be initiated on several hundreds of proteins. To achieve this goal, we will obtain cDNAs either from outside sources or by gene synthesis. To address the challenges associated with producing large amounts of soluble human proteins, we will develop and implement a flexible cloning system for exploring multiple expression vectors. The system is based on the high throughput cloning and expression platform developed during the current Program Project. We will vary the domain boundaries of the targeted genes, test for soluble protein expression, and engineer alternate splice versions. We will further expand the system to utilize fusion proteins, to explore in vitro folding from bacterial inclusion bodies, and to express soluble protein in eukaryotic systems. Soluble proteins will be purified by the most efficient protocols identified.